Synthesis of Complement Components C5, C6, C7, C8 and C9 in Vitro by Human Monocytes and Assembly of the Terminal Complement Complex

Monocytes cultured under serum-free conditions secreted protein which bound covalently and non-covalently to agarose beads, an activator of the alternative pathway of complement. There was a significantly binding of monoclonal anti-C3c antibodies, polyclonal anti-C5, anti-C6, anti-C7, anti-C8, and anti-C9 antibodies, and of a monoclonal antibody against a neoantigen of polymerized C9 to agarose beads incubated with the monocytes for 24, 48, 72 or 96 h. From these results, we conclude that monocytes produce C5, C6, C7, C8 and C9 that assemble as the terminal complement complex on the surface of the agarose beads. Activation by agarose of the alternative pathway with generation of particle bound C3 and C5 convertases is a prerequisite for the subsequent formation of the terminal complement complex. Whether SC5b-9 or the membrane attack of complement (C5b-9) is formed on the beads will be examined.     

Human Umbilical Vein Endothelial Cells Synthesize Functional C3, C5, C6, C8 and C9 In Vitro

Human endothelial cells (EC), cultured serum-free, synthesize de novo protein which increasingly bind to agarose beads (an alternative pathway activator), until a plateau phase is reached after 24-48 h. EC synthesize functional C3, C5, C6, C8 and C9, which were detected on co-cultured agarose beads, using relevant polyclonal anti-complement antibodies. Two monoclonal anti-C9 neoepitope antibodies (aE11, poly C9-MA) bound to the co-cultured beads, showing that the terminal complement complex (TCC) (C5b-9) was assembled on the beads. This also suggests that C7 is synthesized. There seems to be a positive correlation between the amount of agarose-bound labelled protein and agarose-bound complement. The results indicate that EC produce and secrete the components for the functional alternative and terminal pathways of complement.     

Human Alveolar Macrophages Synthesize the Functional Alternative Pathway of Complement and Active C5 and C9 in Vitro

Attachment of protein to agarose beads cultured with macrophages in protein-free medium containing 3H-leucine, shows that de novo synthesis of protein with affinity to the beads takes place. We also found that monoclonal antibodies against human C3c, C3g, and a C9-neoantigen as well as polyclonal antibodies against human C5 and C9, bound to agarose beads that had been kept with the macrophage cultures. Demonstration of C3 derivatives on the agarose beads shows that the essential complement factors of the alternative pathway are synthesized and have been activated by the beads. Deposition of C5 and the detection of a neoantigen of C9 on the beads, indicates that the whole terminal complement pathway has been formed and activated. We conclude that human alveolar macrophages form in vitro the functional alternative pathway of complement, C5 and C9, and we have indirect evidence for synthesis of C6, C7, and C8.     

Human peritoneal macrophages. Production in vitro of the active terminal complement components C5 to C9 and a functional alternative pathway of complement. Brief report

Endotoxin-stimulated human peritoneal macrophages were cultured in serum-free medium with agarose beads. Monospecific antibodies to human C3c, C3g, C5, C6, C7, C8, C9 and to C9-neoantigen bound to the beads. This shows that activated C3 and the terminal complement complex (TCC), made from complement components C5 to C9, were generated on the beads. De novo synthesis was confirmed by agarose binding of tritium-labelled protein. Moreover, C3-derivatives and C9-neoantigen were detected on normal serum-treated agarose beads but not on beads kept in factor B-depleted or heat-inactivated sera, implying that an intact alternative complement pathway was required for our findings. The macrophages thus synthesize the active complement components of the alternative and terminal pathways in vitro.     

Human Alveolar Macrophages Synthesize Active Complement Components C6, C7, and C8 in Vitro

We investigated whether serum-free human alveolar macrophage cultures synthesize active C6, C7, and C8. There was a significant binding of polyclonal anti-human C6 antibodies to agarose beads incubated with unstimulated macrophages for 24 or 48 h. Endotoxin stimulation of the macrophages was necessary for significant binding of polyclonal anti-C7 and anti-C8 antibodies to agarose beads co-cultured for 48 or 96 h. Two monoclonal antibodies (poly C9-MA and MCaE11) specific for a neoantigen of polymerized C9 in the terminal complement complex (TCC), bound to beads mainly incubated with endotoxin stimulated macrophages. The MCaE11 was more sensitive than the poly C9-MA in detecting the C9 neoantigen on beads incubated with the macrophages or human serum diluted 1:16. We thus conclude that human alveolar macrophages synthesize active C6, C7, and C9 that together with C5 and C9, assemble as the TCC on co-cultured agarose beads. Activation of the alternative pathway on the agarose with generation of fixed C3 and C5 convertases is a prerequisite for the subsequent generation of the TCC.     

Formation of the Terminal Complement Complex on Agarose Beads: Further Evidence that Vitronectin (Complement S-Protein) Inhibits C9 Polymerization

Vitronectin occupies the metastable binding site of C5b-7, which is unable to insert membranes as part of the complement lytic attack. Some evidence has been presented that vitronectin inhibits also membrane-associated pore formation by inhibiting C9 polymerization in the terminal complement complex (TCC). The authors wished to add to this background by studying the effect of vitronectin on formation of TCC on a carbohydrate surface like agarose beads, an alternative complement pathway activator. Bound TCC was detected by monoclonal and polyclonal antibodies to C9-neoepitopes. Soluble SC5b-7 and TCC (SC5b-9) did not bind to the agarose beads. Using serum or isolated complement factors for the alternative and terminal pathways, the authors found that vitronectin reduced the density of C9-neoepitopes on the beads. As there was no convincing evidence for association of vitronectin with the factors C5b-8 of the agarose-bound TCC, it was concluded that vitronectin bound directly to C9 in TCC and inhibited C9 polymerization within the complex. The authors have shown that TCC can bind to a carbohydrate surface like agarose (an alternating polymer of galactose moieties) in the absence of lipid. These results suggest that vitronectin can limit the lytic effect of membrane-bound TCC by inhibiting C9 polymerization.     

Synthesis of Soluble C3 and C9 Neoepitopes by Human Alveolar Macrophages in Vitro

The aim of this study was to examine whether soluble neoepitopes of activated C3 (C3b, iC3b, C3c) and C9 are produced by human alveolar macrophages cultured in serum-free medium. There was a significant and inhibitable production of C3 and C9 neoepitopes and C9 by the macrophages from all donors, as detected by enzyme-linked immunosorbent assays based on monoclonal (bH6, aE11) and polyclonal (anti-C9) antibodies. A strong donor-dependent variation in the levels of the C3 neoepitope and C9 (five- to sevenfold) and the C9 neoepitope (twofold) was found. After 1 day (24 h) of incubation, the complement levels were largely unaltered. The presence of an exogenous alternative pathway activator (agarose beads) reduced the amount of soluble complement because of binding to the agarose. However, the relative fraction of C9 neoepitope versus C9 increased (two- to threefold), due to agarose-mediated activation of C9. The results demonstrate activation of the complement system in serum-free alveolar macrophage cultures, irrespective of the presence of a known complement activator.     

Formation of the Functional Alternative Pathway of Complement by Human Monocytes in Vitro as Demonstrated by Phagocytosis of Agarose Beads

Native agarose beads (diameter 5-10 micron), activators of the alternative complement pathway, are slowly phagocytosed when incubated with human monocytes cultured under serum-free conditions. Agarose beads preincubated with monocyte cultures and then transferred to new cultures are more easily phagocytosed than native beads. These results indicate that the phagocytosis of agarose beads depends on opsonization of the beads by one or several substances of monocyte origin. By using antihuman C3 antibodies, trypsin treatment, and sodium dodecyl sulphate washing, we were able to demonstrate C3b and iC3b on the agarose beads. The molecules were covalently bound to the surface of the beads. We conclude that in vitro human monocytes produce and secrete the essential factors for activation and propagation of the alternative complement pathway (factors C3, B, D, H and I), which becomes evident with an external activator like agarose beads in the cultures. The activation of complement by agarose beads results in the attachment of C3b and iC3b to the surface of the beads, which are then phagocytosed by means of C3b and iC3b receptors on the monocytes.     

Synthesis of Complement by Alveolar Macrophages from Patients with Sarcoidosis

Sarcoidosis is a granulomatous disorder of unknown aetiology. Alveolar macrophages (AM) in sarcoidosis release a variety of mediators important to the pathogenesis of the disease. Complement is essential for the inflammatory response and we investigated whether there were any major defects in the potential for sarcoidosis AM to synthesize complement in vitro. AM from 11 patients with active sarcoidosis and three healthy controls were cultured under serum-free conditions. There was a significant binding of polyclonal (anti-C5, -C6, -C7, -C8) and monoclonal anti-complement antibodies (anti-C3c and anti-C9 neoepitope (aE11] to agarose beads incubated with unstimulated AM for 24, 48, or 72 h. A significant and inhibitable production of soluble C3c, C5, C9, and S-protein was found in the harvested medium as detected by enzyme immunoassays. Activated C3 and C9 were also detected based on neoepitope expression. Presence of co-cultured agarose beads reduced the amount of soluble S-protein due to deposition on the agarose. We argue that the C9 neoepitope is an integral part of the terminal complement complex (TCC), both in the fluid and solid phase when bound to the agarose. In the fluid phase, SC5b-9 was generated, whereas the agarose-bound S-protein is assumed not to be associated with TCC on the beads. The results demonstrate for the first time that AM from sarcoidosis patients synthesize the functional alternative and terminal pathway of complement.     

Attachment and phagocytosis by salmon macrophages of agarose beads coated with human C3b and C3bi

Agarose beads (diameter 5-10 micron) preincubated in human serum became associated (attached and ingested) to 50-60% of the salmon macrophages within 60 minutes. However, Beads preincubated in serum treated with heating (50 degrees C, 20 min) or with EDTA (10 mM) to inhibit the activation of alternative complement pathway, were not associated to the phagocytes. Furthermore, agarose beads coated with human C3b and C3bi after incubation with isolated complement factors (C3, D, B), were associated to 30-40% of the phagocytes. About 80% of the cell-associated agarose beads was intracellularly located. Conversion by trypsin treatment (0.01%) of agarose bound C3bi to C3d, abolished the association of such beads to the macrophages. The results demonstrate that salmon macrophages possess complement receptors that bind human C3b and C3bi. Agarose beads coated with these ligands (C3b and C3bi) are attached and ingested by the phagocytes.     

Immunohistochemical Detection of the Membrane and Fluid-phase Terminal Complement Complexes C5b-9(m) and SC5b-9

Activation of the terminal pathway of complement on a membrane results in the generation of the membrane-damaging terminal C5b-9(m) complement complex, whereas the non-lytic water-soluble SC5b-9 complex is formed when complement is activated in the fluid phase. Both forms of the terminal complement complex (TCC) can be immunohistochemically detected, but not distinguished, by antibodies recognizing neoantigens in the complexes. By means of monoclonal antibodies against C9 neoantigens and against the S-protein, it was demonstrated that deposits of the TCC in tissue sections may be either in the form of C5b-9(m) or SC5b-9. The consequences of this for the interpretation of the histochemical data and the terminology of the two complexes are discussed.     

Phagocytosis of Agarose Beads by Receptors for C3b (CR1) and iC3b (CR3) on Alveolar Macrophages from Patients with Sarcoidosis

Alveolar macrophages (AM) from sarcoidosis patients exhibit no detectable defect in their potential to synthesize the functional alternative and terminal pathway of complement. They also synthesize more C9 than AM from healthy controls. Various authors [4, 6] have suggested that sarcoid AM have decreased phagocytic ability. In the present work we studied whether there was any difference in C3 receptor-mediated phagocytosis of serum-treated and native agarose beads by AM recovered from patients with active sarcoidosis compared with controls, AM from seven patients with active sarcoidosis and seven healthy controls were cultured under serum-free conditions for 2, 12, 24. and 48 h. We found a significantly increased CR1 and CR3 receptor-mediated phagocytosis of native agarose heads by AM from the seven patients. CR1 and CR3 were also detected on AM directly recovered from bronchoalveolar lavage fluid using fluorescein-conjugated monoclonal anti-receptor antibodies. The percentage of AM expressing CR appeared to be increased in sarcoidosis. The reason for the enhanced phagocytosis of agarose beads by the sarcoid AM is probably the result of both increased synthesis and receptors of complement. Altered complement production and complement receptors may be important for the pathogenesis of this granulomatous disorder.     

Extracellular phosphorylation of C9 by protein kinase CK2 regulates complement-mediated lysis

Ecto-protein kinases (ecto-PK) are expressed on many cell types, both normal and malignant, yet their functions are largely unknown. An ecto-PK capable of phosphorylating the C9 component of the complement system is described. This C9 ecto-PK could be inhibited by TBB, Emodin and DRB, selective inhibitors of protein kinase CK2. Treatment of Raji human B lymphoma cells with these CK2 inhibitors augmented cell killing by Rituximab (anti-CD20 antibodies) and human complement. Analysis of C5b-7-bearing Raji cells showed that extracellular inhibition of the ecto-CK2 enhanced cell lysis by C8 and C9. Blocking of the membrane complement regulator CD59 with monoclonal antibodies further enhanced the effect of the CK2 inhibitors on Raji cell death by complement. C9 ecto-CK2 activity was increased on cancer cells relative to normal fibroblasts and blood cells. Therefore, ecto-CK2 appears to be an additional factor protecting cells from complement-mediated lysis, probably by phosphorylation/inhibition of complement C9.     

Complement activation in septic baboons detected by neoepitope-specific assays for C3b/iC3b/C3c, C5a and the terminal C5b-9 complement complex (TCC).

We have investigated the cross-reactivity of various species in neoepitope-specific methods for quantification of human complement activation products. In contrast to most other species examined, baboon showed a substantial cross-reactivity supporting a high degree of homology between human and baboon complement. An assay for C3b, iC3b and C3c (MoAb bH6) showed moderately good reactivity, in contrast to a C3a assay which did not cross-react. Excellent reactivity was found for C5a using MoAbs C17/5 and G25/2. The reactivity of an established TCC assay (MoAb aE11 to a C9 neoepitope and polyclonal antibody to C5) was improved substantially by replacing the anti-C5 antibody with a new MoAb to C6 particularly selected on the basis of baboon cross-reactivity. Plasma samples from baboons receiving 2.5 x 10(9) and 1.0 x 10(10) live Escherichia coli bacteria/kg were examined with the assays described. In vivo complement activation with the lowest dose was moderate and kept under control, in contrast to the highest dose, where an uncontrolled increase in all activation products continued throughout the infusion period. These results support the hypothesis that sufficiently high amounts of endotoxin lead to uncontrolled activation of complement as seen in irreversible septic shock. The results are discussed with particular emphasis on activation of the terminal complement pathway.     

Regulation of complement activation at the C3-level by serum resistant leptospires

Leptospires are spirochetes that are transmitted to humans through contacts with wild or domestic animals or via an exposure to contaminated soil or water. In this study we have compared the serum-sensitivity of five pathogenic strains of leptospires (L. interrogans) to an environmental isolate (strain Patoc). Different levels of sensitivities to human serum were seen. Interestingly, the most sensitive strain was the non-pathogenic Patoc strain. The fully and intermediately resistant strains have been isolated from human patients. Testing was performed in the absence of specific antibodies, and killing was found to be dependent on the complement system. The serum sensitive Patoc strain was killed in human serum within minutes, whereas the most resistant strains tolerated serum up to 4h. We also tested the deposition of the complement components C3, C5, C6, C8 and C5b-9 to the surfaces of the sensitive and resistant strains of Leptospira by immunofluorescence microscopy and ELISA. C3 was deposited on both the sensitive and resistant strains, but the terminal complement components were detected only on the surface of the complement-sensitive strain. The complement resistant and intermediate strains were found to bind more factor H from human serum than the complement sensitive strain. Thus, binding of this major alternative complement pathway inhibitor is related to serum resistance in Leptospira spirochetes.     

Stimulation of Ca2+-dependent chemiluminescence in rat polymorphonuclear leucocytes by polystyrene beads and the non-lytic action of complement.

(1) Chemiluminenscence of rat polymorphonuclear leukocytes was stimulated by a phagocytic stimulus, latex beads (diameter = 1.01 micrometer). The maximum chemiluminescent intensity increased with bead concentration in the range 0.2--20 x 10(9) beads/ml. This response was abolished in the absence of extracellular Ca2+ (1 mM EGTA). (2) Chemiluminescence could also be stimulated by the Ca2+ ionophore A23187 in the presence of extra-cellular calcium. (3) Addition of human serum, as a source of complement, to rat polymorphonuclear leukocytes preincubated with anti-5''-nucleotidase serum resulted in a rapid stimulation of chemiluminescence, after a lag of about 40 s. (4) The stimulation of chemiluminescence by antibody plus complement was not the result of cell lysis because (i) no significant release of lactate dehydrogenase was detected at the time of the chemiluminescent response (ii) chemiluminescence was associated with the cells and not the surrounding media (iii) cell lysis did not produce chemiluminescence. (5) Chemiluminescence stimulated by antibody plus complement or by beads was inhibited by the ''calmodulin-blocker'', trifluoperazine (50% inhibiton with approximately 20--30 microM). (6) Cu2+ (10(-4) M), which can inhibit C9 action, inhibited the rapid rise in chemiluminescence induced by antibody plus complement, but not the bead-induced chemiluminescence. (7) Depletion of C9 from human serum markedly inhibited the complement induced chemiluminescence response. Addition of purified C9 restored the response. (8) It was concluded that formation of the terminal complement attack complex at the surface of rat polymorphonuclear leucocytes induces a Ca2+-dependent chemiluminescence in the cells, in the absence of cell lysis.     

The use of flow cytometry to detect the biosynthesis of complement components

Agarose beads, an activator of complement, were incubated with MRC-5 or He 9 fibroblast cell lines under serum-free conditions. The beads were tested for binding of anti-complement antibodies by flow cytometry with a FACS 440 using FITC-labelled anti-Ig detection antibodies. Controls consisted of co-cultured beads incubated with irrelevant antibody or albumin, beads maintained in cell cultures containing cycloheximide, and beads which were not exposed to cells. The histograms demonstrated positive staining with anti-C3c, -C5, -C7 and -C9, but not with anti-C6 and -C8. Flow cytometry with multiple histogram analysis confirmed that the differences between the positive curves and the controls were statistically significant. The results show that cell-derived complement components (C3, C5, C7 and C9) were deposited on the beads and could be detected by flow cytometry.     

Quantitation of activation of the human terminal complement pathway by ELISA

We have devised an enzyme-linked immunosorbent assay (ELISA) to quantitate fluid phase terminal complement pathway activation. Upon activation to form C5b-9, terminal complement components express neoantigens not present in the unassembled individual components. Expression of one of these neoantigens occurs at the step of C9 activation. C9 neoantigen is present in fluid phase SC5b-9 complexes, membrane-bound MC5b-9 complexes, and in in vitro polymerized C9. Under physiologic conditions, the presence of C9 neoantigen indicates that the terminal complement pathway is activated through the terminal component C9. In our assay for C9 neoantigen, we used rabbit antiserum to polymerized C9 rendered specific for C9 neoantigenic determinants by serial absorption with human serum, human C9, and other terminal complement components bound to Sepharose. Using the IgG from this antiserum, we devised a sandwich ELISA to bind SC5b-9 from solution onto polystyrene plates. The ELISA plates were developed with the use of goat antiserum to native C9 epitopes followed by a swine anti-goat IgG-alkaline phosphatase conjugate. Quantitation of SC5b-9 in solution was performed by comparing sample OD to a standard curve generated with human SC5b-9 that was purified from zymosan-activated serum. The assay was sensitive to as little as 100 ng of SC5b-9/ml and should be useful for screening plasma, serum, cerebrospinal fluid, or other biological fluids for the presence of terminal complement pathway activation.     

Immunohistochemical Localization of C9 Neoantigen and the Terminal Complement Inhibitory Protein CD59 in Human Endometrium

PROBLEM : Human endometrium expresses complement components, receptors, and regulatory proteins, many of which appear to be expressed in a hormone-dependent manner. Whether terminal complement components are also present in the endometrium is unknown. CD59, a broadly expressed protein that blocks association of C9 with C8 in the membrane attack complex, is localized in reproductive tissue to human spermatozoa, seminal plasma, amniotic fluid, and placenta. The present study examines human endometrium for the presence of CD59 and terminal complement proteins. METHOD : Endometrial biopsies were obtained from six normal women from various phases of the menstrual cycle and analyzed by immunohistochemistry, using MEM-43 anti-human CD59 and anti-human SC5b-9 murine monoclonal antibodies and the immunoperoxidase technique. RESULTS : Both CD59 protein and SC5b-9 (C9 neoantigen) were demonstrated to be present in endometrial glandular epithelium throughout the menstrual cycle. No specific staining was demonstrated in the stromal compartment. CONCLUSION : CD59 protein and terminal complement proteins are expressed in glandular epithelial cells of normal human endometrium, in both proliferative and luteal phases, suggesting that expression is not hormonally dependent. These analyses further support the presence of a functionally active complement system in normal human endometrium.